1 Section of Cell Biology, Laboratory of Cellular Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America, 2 Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America

Abstract

Introduction

XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.

Methods

Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.

Results

MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.

Discussion

The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.

Editor: K.T. Jeang, National Institute of Health, United States of America

Received: October 28, 2011;

Accepted: January 31, 2012;

Published: March 16, 2012

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: SSR is a recipient of a postdoctoral fellowship at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an intra-agency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.* E-mail: chintamani.atreya@fda.hhs.gov# These authors contributed equally to this work.

This article is very interesting for several reasons. First, it demonstrates again that XMRV will infect multiple human cell types. Two of these, cell lines LNCaP and DU145, are described as robustly infected, while ordinary peripheral blood leukocytes (PBL) and monocyte-derived macrophages (MDM) are only moderately infected. Specific cancer cells were more vulnerable to infection than immune cells isolated from peripheral blood. All these cell types are highly relevant to the controversy, and these are unquestionably human cells, not mouse cells. (The nude mice in which XMRV is postulated to have originated do not even have some classes of leukocytes, thus do not provide an evolutionary environment for some adaptations.) .

Second, microRNA is important in gene expression found in several oncogenic viruses: reticuloendotheliosis virus (REV), EBV and HCV. REV is a gamma retrovirus. This shows this retrovirus, XMRV, is interacting with gene expression in target cells in ways relevant to pathology. From an evolutionary standpoint, differences in gene expression are even more important in distinguishing human cells from other species than differences in genes coding for proteins. The bulk of the differences between human and chimpanzee genomes are in the regulation of genes, not the proteins produced. Differences between human and mouse genomes would appear to be more pronounced. It is difficult to see how this virus rapidly acquired these adaptations in a mouse environment devoid of human immune cells, and with defective immune response.

In addition to actual cancer cells this study found infection altered functioning of hematopoietic cells. This study fits in with independent findings of immune system involvement in pathogenesis of ME/CFS and several other diseases. The list of coincidences blamed on an improbable series of chance recombination events taking place in a short time in a single mouse keeps getting longer.

Specific concentration on a single sequence has probably been an error. It is unlikely to be the unmodified product of a simple infection. However, something else with similar characteristics should be highly suspect.

Unlike many here I am not in favor of the vaccine contamination hypothesis. Contamination does occur, but it produces characteristic patterns I don't see. What I keep seeing is a response by immune systems which are already compromised to ANY immune challenge. Experience in troubleshooting complex technological systems has made me very aware of the difference between a defect or fault and the conditions which expose that fault.

If you find this idea weird, consider those pieces of software you buy, or rather license. All the bugs that turn up later (except those introduced by later attempts at correction) are already present in the DVD you get when you buy the program. (I'm assuming you are using the antiquated system of buying software from a brick-and-mortar store, or at least ordering it on-line, to be installed from a physical medium.) The apparently unending cascade of updates results from exposure of those bugs. The genius of Bill Gates lay in getting people to pay him for bugs, which everyone else kept trying to avoid.

Contamination also occurs in non-biological contexts where I've seen it. Tracking the source can be maddening, but you start to put together patterns which reveal that source. A great deal of attention is already focused on possible and plausible sources of contamination. What I'm seeing, if it is contamination, is more like a source nobody has a clue about.

As an example consider the spread of HIV-1 in Africa following an assumed origin sometime around 1910. A great deal of attention has focused on vaccines, because that is where we have the most documentation. What is missing from all documentation is any record of how many times unsterilized needles were reused during vaccination drives. Inadequate funding and corruption meant that health professionals on the lowest levels were often faced with such limited resources that reusing needles was the only way to meet vaccination schedules and budgets. At the time nobody thought the reused needles could be carrying an unknown disease more deadly than polio or smallpox, because the patients were asymptomatic for years. If highly-trained medical researchers discounted that possibility you can imagine how people with inadequate training acted. All concerned were blindsided by the possibility they did not consider or document, though only some lost their lives as a result. Professional blindspots are precisely what I'm looking for.

What has become increasingly apparent to me is that the assumed lack of active human retroviral infections, other than HIV and HTLV, is not necessarily true. In the years before anyone knew about viruses, to say nothing about retroviruses, it is certain that virtually the entire human race was constantly exposed to retroviruses which have repeatedly shown the ability to jump species. Many examples in domesticated animals would be unlikely to have been propagated far prior to domestication because the different species would seldom interact the way they do under human control. The human retroviral environment of the last 10,000 years has been radically different from the previous several million years. Molecular clock arguments have not caught up with this change.

After trying to make sense of the interplay between ERVs and exogenous retroviruses, as depicted in scientific literature, I came to the conclusion that infections that began before modern medicine, and were propagated from parent to offspring, would explain several medical mysteries -- provided human hosts were generally capable of holding a retrovirus latent for long periods of time. This would also invalidate a number of arguments convenient in research. An infection inherited in the perinatal period, when infants are immunologically naive, and lasting most of a lifetime, would confound quite a number of attempts to compare sick individuals with "healthy controls". Retroviral infections could also make hash of a number of genetic studies, which typically assume all genes arrived by Mendelian inheritance.

Until we have thoroughly compared the ERVs in all domesticated species with HERVs we will not know if horizontal transmission has occurred in the last few thousand years. Even ERVs which can be traced back millions of years may have active close relatives, as in the case of HERV-Fc1. Viruses which infect domesticated animals can jump to humans, particularly humans who consume animal products like meat or milk. Viruses which infect humans can certainly jump to animals which thrive in areas filled with waste products of human civilization. Jumping back is quite possible.

What seems increasingly unlikely to me is the currently dominant medical attitude that humans are uniquely invulnerable to most retroviruses around them. I think humans are often infected, but mostly control these infections until other factors compromise immune systems. This means that some infectious diseases present to doctors in contexts where it is especially hard to isolate the original pathogen. (I'm not even talking here about the special diagnostic confusion in the case of ME/CFS. For that consider how hard it would be to isolate varicella zoster virus if you weren't allowed to test patients for six months after they came down with chickenpox.)

This reasoning would then apply to a number of diseases where progress on etiology, prevention and cure has been at a standstill for about 50 years. I particularly suspect undetected gamma and beta retroviruses, because of the number of reports of similar sequences and retroviral activity in humans. What I do not expect is that such retroviruses will remain unchanged or behave identically to retroviral infections in other species. We are apparently unique in the extent to which we have domesticated other species, we would not have survived as a species if we did not have good adaptations to this peculiar situation. This does not say that all members of the species are invulnerable, or that any member is invulnerable for life.

The micro RNA described in this paper can downregulate or even silence particular genes. RNA interference would provide precisely the missing mechanism needed for holding inserted provirus latent, even when proviral genes are transcribed into mRNA. I think we have just gained a glimpse into another battleground between retroviruses and hosts.

Just to clear up any confusion about what the study was about: this study focused on the expression of human miRNAs in cultured cells infected with XMRV. "micro RNAs" (miRNAs) are given that name because they're very short sequences of RNA. Our DNA codes for them and, although they don't code for proteins themselves, they serve all kinds of functions in our cells. Mostly they function by essentially up or down-regulating genes (usually the latter) as needed. Some appear to have an anti-viral function, interfering with viral replication. Some viruses, it turns out, code for their own microRNAs, which serve to help these viruses increase their replication/infective efficiency.

A number of viruses that cause cancer have been shown to do so, at least in part, by changing the expression of host miRNAs that are important for cell division/growth. The 'oncogenes' that are activated include miRNA encoding segments that, if switched on or off in the wrong way, can lead to tumor growth. This study looked at human miRNAs, and whether there was evidence that XMRV was regulating them.

They found that in XMRV-infected cells, a number of miRNAs were being expressed differently than in control (uninfected) cells. Two of these were common to all four cell types they studied. One of these two miRNAs is predicted to impact five genes related to tumor formation or suppression (one of those five genes, interestingly, is a gene that in mice is also regulated by Friend MLV, which favors it as an integration site).

A question one could raise in light of this study: Do cells co-cultured with CFS patient blood exhibit the same or similar patterns of miRNA expression as those experimentally infected with XMRV (or at least different patterns than controls)? If it did, it couldn't at this point be taken to indicate retroviral infection, but it would still be very interesting.

These authors also found that miRNA expression patterns differed between prostate cells and blood-derived cells (lymphocytes and macrophages) infected with XMRV. Changes in the expression of a given miRNA can have very different results in different cell types. I did a brief google search on miR-142-3p, one of the miRNAs listed in Figure 5 that was differentially expressed in the blood cells but not in the prostate cells; turns out its overexpression is linked to cancer in T-cells, appears to suppress a gene that favors cancerous invasion in liver cells, and - most interestingly - is involved in endotoxin-induced IL-6 production in dendritic cells. IL-6 is one of its targets, in fact, according to Sun et al 2011 (http://bloodjournal.hematologylibrary.or...abstract).

That's especially interesting as retroviruses have been known to upregulate IL-6, and IL-6 is one of the cytokines reported to be upregulated in CFS patients (including in both the Klimas and Mikovits cytokine profile studies).

Thanks anciendaze and Dr. Yes. it sounds like a whole new frontier in possibly furthering our understanding of many chronic and baffling illnesses. We have used the word autoimmune to describe so many illnesses, but the term is not a very satisfying answer without any real understanding of why the process began.

I hope there is much more activity going on down this avenue of research. There is a chance to effect a paradigm shift in medicine.

Thanks anciendaze and Dr. Yes. it sounds like a whole new frontier in possibly furthering our understanding of many chronic and baffling illnesses. We have used the word autoimmune to describe so many illnesses, but the term is not a very satisfying answer without any real understanding of why the process began...

Elsewhere, I've pointed out that various types of immune memory cells hold antigens for which the body may need to produce antibodies in the future. In the case of some viruses, particularly retroviruses, the "memory" might well be the virus itself held latent in that cell. From this viewpoint, B-cell depletion, for those cells with the CD-20 epitope indicating it is on the case, might do more than simply reset the "memory" -- it might clear the body of infection to the point where other immune cells can gain the upper hand. This could be what B-cell depletion with Rituximab is doing in several autoimmune disorders.

Learning that one virus, XMRV, can downregulate production of hypermutation enzymes, and a mechanism by which this is done, sounds useful. Learning that host cells can silence genes inserted by retroviruses at the stage of mRNA is likewise a breakthrough. This is quite an accomplishment for a virus whose study has been called "a waste of money". A useful discovery opening new research paths might even end all the fingerpointing about what went wrong in the past.

Pragmatism involves accepting things that are useful without getting too worked up over the truth of earlier claims. The antibiotic properties of sulfanilamide were found through such a fruitful mistake. The dye molecule which turned patients red added nothing to the medicinal properties of the raw material.

I've put together some material on the range of infective diseases caused by retroviruses in domesticated animals: leukemia/lymphoma, adenocarcinomas (of both ducted glands and the lungs), sarcomas, immune deficiencies, neurodegenerative diseases, arthritis. You might compare this with a list of human diseases of unknown etiology. It would be foolish to claim humans are not exposed to these viruses.